Extracellular ATP regulates many physiological processes via release of nitric oxide (NO). ATP stimulates NO in thick ascending limbs (TALs), but the signaling cascade involved in the cells of this nephron segment, as well as many other types of cells, is poorly understood. We hypothesized that ATP enhances NO synthase (NOS) activity by stimulating PI3 kinase and Akt. We measured a) NO in TALs using the NO-sensitive dye DAF-2 DA and b) Akt activity by fluorescence resonance energy transfer (FRET) and phosphorylation of Akt isoforms. ATP (100 µM) stimulated NO in wild-type mice (26±4 arbitrary units, AU), but not in NOS3 -/- mice (2±2 AU; p<0.04). In the presence of the NOS1 and NOS2 selective inhibitors 7-NI and 1400W, ATP stimulated NO by 30±2 AU and 33±3 AU, respectively (N.S. vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5±2 vs. 28±4 AU; p<0.02). ATP alone increased Akt activity and this effect was significantly blocked by suramin, a P2 receptor antagonist. In the presence of an Akt-selective inhibitor, ATP-induced NO was blocked by 90±4%. ATP significantly stimulated Akt1 phosphorylation at Ser⁴⁷³ by 91±13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominant-negative Akt1 significantly decreased ATP-induced NO by 88±6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL.